The substructure of tight and gap junctions is investigated by direct freezing techniques that avoid any chemical fixation and serve to increase the definition of individual membrane components. A new model of tight junction structure was developed which replaced the previous view these junctions are comprised of rows of intramembrane proteins; rod-shaped structures seen after direct freezing are now interpreted as inverted cylindrical micelles of membrane lipids. Evidence for this model has been gathered from investigations of pure lipid bilayer systems which are induced to form non-planar micellar phases by addition of calcium ion. Cylindrical lipid micelles identical to the cylinders at tight junctions are found embedded in these lipid bilayers. Tight junctions, but not septate junctions, in invertebrates also appear to have lipidic backbones. How tight junctions prevent small charged solutes from entering the brain (across the blood-brain barrier) is made clear by this new model of tight junction structure. Gap junctions form within minutes of treating cells with certain mild lipid solvents, suggesting that precursors of the intramembranous components of gap junctions are continuously present in the cell membrane. Direct freezing techniques were also used to investigate the distribution of endothelial vesicles and other intracellular organelles in capillaries that had not been subjected to chemical treatment or fixation. Approximately threefold fewer apparently free vesicles were found in directly frozen endothelial cells, as compared with aldehyde-fixed cells, from capillaries of the eel swim bladder and also from cultured human endothelium. However, ultrathin serial sectioning showed that in both cryofixed and chemically fixed eel capillaries, as in the capillaries of the frog mesentery, virtually all vesicles are in continuity with the plasma membrane.